Selective removal of albumin from blood fluids and compositions therefore

ABSTRACT

Certain compositions have been prepared by the reaction of certain dyes of the class generally known as Color Index reactive dyes with certain support phases. The dyes utilized for said compositions all possess the general sulfanilido-triazidinyl-sulfoaryl group wherein the aryl groups may be phenyl or naphthyl and said general group is further bonded to aryl groups via an amino or azo linkage. It has been found that when aqueous fluids containing albumin, such as, for example, plasma or serum are passed over compositions within the scope of the present invention albumin is selectively adsorbed on said compositions without removal of other proteins present in the fluid. It has further been found that the albumin may be readily removed from the thus formed adsorbate without denaturing the albumin and leaving the solid phase dye composition in a suitable state for re-use without loss of activity.

The invention described herein was made in the course of work undergrant or award from the Department of Health, Education and Welfare ofthe United States of America.

RELATED APPLICATIONS

This application is a continuation-in-part of applicant's co-pendingapplication, Ser. No. 601,676, filed Aug. 4, 1975, now U.S. Pat. No.4,016,149, which in turn is a continuation-in-part of their thenco-pending application, Ser. No. 396,036 filed Sept. 10, 1973, nowabandoned.

FIELD OF THE INVENTION

Selective fractionation of proteins in blood fluids.

DESCRIPTION OF THE PRIOR ART

It is well known that blood fluids such as plasma and serum contain manyproteins which it is desirable to isolate in a pure state. Isolationprocedures for such proteins are known, however, they all suffer from acommon problem, namely, the presence of albumin. Albumin is the majorprotein constituent in plasma. Besides being a contaminant in theaforementioned procedures, it is often desirable to isolate pure albuminuncontaminated by other proteins. Thus, it would be most desirable todevelop a procedure whereby albumin can be selectively removed fromblood fluids without simultaneous removal of other proteins which may bepresent in very small quantities.

It is well known that certain proteins will bind very tightly to certainreactive dyes. It has been reported that Blue Dextran will bind tightlywith lysozyme, ovalbumin, and hemoglobin when they carry net positivecharge, and bovine serum albumin independent of charge. Blue Dextran isa dye generally known as Procion Blue HBS wherein the chlorine atom onthe triazine moiety of the dye is replaced by the polymer Dextran beingbonded to said triazine group via --O-- grouping. (White and JencksBinding of Proteins to Blue Dextran, No. 43, Biol., Abstracts, 160thNational Meeting, American Chemical Society, September 14, -18, 1970).It should be noted however that Blue Dextran is water soluble. Thus, anoperative procedure would involve coupling Blue Dextran to a solidsubstrate from which the albumin can be removed. It would beparticularly desirable if, in addition to removal of the albumin in sucha manner that the absorptive capacity of the substrate was not damaged,the albumin was regenerated in such way that it was not denatured.

Cuatrecasas has disclosed a method of protein purification by affinitychromatography. In the Cuatrecasas procedure insoluble materials areprepared by coupling certain large molecules containing amino groups toagarose by activating the agarose with cyanogen bromide prior totreating the activated agarose with the amino containing moiety. (J.Biol.Chem. 245,3059,(1970).

Bohme et al (J. Chromatogr., 69, 209, (1972)) found that thecondensation product of Cibacron Blue F3G-A, otherwise known as ProcionBlue HBS with Dextran 2000, otherwise known as Blue Dextran 2000 (atrademarked product of Pharmacia, Uppsala, Sweden) had a high affinityfor the enzyme phosphofructokinase. On the other hand, the condensationproduct of Dextran 2000 with Cibacron Brilliant Blue FBR-P, a dye ofextremely similar structure, showed no affinity for phosphofructokinase.Neither was such activity shown when Blue Dextran was coupled withcyanogen bromide activated sepharose.

The specificity of dyes for certain polypeptides is therefore notpredictable.

SUMMARY OF THE INVENTION

It has been found that certain reactive dyes when coupled to certainsolid support phases provide a composition upon which albumin may beselectively adsorbed out from the presence of other proteins fromsubstantially cell free aqueous fluids such as plasma or serum. Theadsorbed albumin may be removed, in high yield, without denaturization,from said compositions and compositions may be re-utilized without lossof adsorbative capacity. The support phases which may be utilized forthis purpose include agarose, more particularly sepharose,polyacrylamides, more particularly cross-linked polyacrylamide gel, andacrylic resins, more particularly anion exchange resins such asAmberlite IRC 45, containing an amino group.

These support phases are bonded to Color Index reactive dyes(hereinafter CRD) wherein the CRD has the general formula ##STR1##wherein R₁ is hydrogen, an alkali metal, or an alkaline earth metal, R₂is halo or --O-- dextran

R₃ is hydrogen, alkyl, phenyl, alkoxy phenyl, alkyl phenyl and ##STR2##Z₁ is [-H]₂ or --CH═CH--CH═CH-- Z₂ is a nitrogen-nitrogen bond or ahydrogen attached to the nitrogen shown only,

Q is substituted or unsubstituted aryl where Z₂ is hydrogen orsubstituted or unsubstituted aryl-N═ where Z₂ is a double bond, providedthat the substituent groups aforesaid are other than halo or heavy metalwherein heavy metal is defined as a metal falling in periodic groupsother than 1 or 2A, m is 1 or 0 and n is 1 or 2.

The adsorption of the albumin onto the support phase/CRD from the fluidcontaining it, is suitably carried out in a buffer of at least 0.05Mmolarity at a pH of between 6.5 and 8.5. While the process of theinvention is not limited thereto and any method of contacting isconsidered within the scope of the invention, it is consideredconvenient to charge the suspension of the support phase/CRD compositionto a chromatographic column and to pass through said column, thesubstantially cell free albumin containing fluid. The eluate issubstantially albumin free and may be further processed as desired.

The albumin is then removed from the support phase/CRD suitably byeluting, preferably eluating a column, with a suitable eluent. Theeluents may be selected from the group consisting of Chaotropes such as:aqueous Urea, aqueous quanidino-hydrochloride, buffered aqueous, loweralkanols, suitably buffered aqueous ethanol and aqueous thiocyanate inassociation with any cation giving rise to sufficient solubility.Alkanoic acids suitably alkaline buffered alkanoic acids containing 2 to10 carbon atoms and aqueous solutions containing certain cations,suitably those of potassium and the group 2A metals. The use of any ofthese eluents will provide a support phase/CRD composition of renewedactivity which may be used to remove further amounts of albumin fromadditional charges of albumin containing fluids. However, where it isdesired to isolate pure, nondenatured albumin the latter two groups ofeluents, as well as aqueous thiocyanate solution should be employed. Thealbumin can then be isolated therefrom by, say, lyophilization in theusual manner.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one embodiment of the present invention there may be employed assupport phase agarose, preferably in the form of sepharose which may bein the form of packed sepharose (for example grades 2B, 4B or 6B) orcross-linked sepharose (for example, grades 2B, 4B, or 6B). As statedheretofore, the reactive dyes which may be utilized in the presentinvention possess certain common structural characteristics. Certain ofthese dyes may be more particularly identified by means of their ColorIndex generic name, Color Index constitution number, or commercial name.Among these dyes thus identified may be included the following: CIReactive Orange 2 (CI Constitution No. 17865, also known as CibacronOrange G-E, or Procion Brilliant Orange HGRS), CI Reactive Red 4 (CICN18105, also known as Cibacron Brilliant Red 3B-A, or Procion BrilliantRed H7BS), CI Reactive Brown 1 (CICN 26440, also known as Cibacron Brown3GR-A, or Procion Orange Brown HGS, CI Reactive Red 9 (CICN 17910, alsoknown as Cibacron Scarlet 2G or Procion Scarlet H3GS). Of similarstructure is CI Reactive Red 16, also known as Cibacron Scarlet 4G-B.Also included in this group are the anthraquinone dyes CI Reactive Blue2 (CICN 61211, also known as Cibacron Blue F3G-A or Procion Blue HBS,and CI Reactive Blue 5 (CICN 61210, also known as Cibacron BrilliantBlue FBR-P).

Methods of making all of these dyes will be found in Pancharteck, etal., Col.Czech.Chem.Commune., 25, 2783 (1960).

Also within the scope of the present invention should be consideredcompounds wherein the terminal benzene sulfonic acid group is replacedby another substituent such as alkyl, phenyl, alkyl phenyl, alkoxyphenyl, hydrogen or the like. Typical of this group is CI Reactive Red(CICN 18159) (C. Calin, et al, Coloristica Buletin Informativ, 4, (No.11, 1968) 179-200.

All of the foregoing dyes may be coupled with the support phasesubstrates by heating in water at moderately elevated temperatures,suitably above pH 6.0. In the preferred modification the mixtures areheated with stirring between 75° and 95°, the mixture cooled, and washedto remove the unbound dye.

The ratio of the weight of dye to the weight of support phase is notcritical. Where too little dye is utilized the efficiency of theextractive process will be reduced in view of the presence of too fewadsorption sites. If too much dye is used the product obtained is notonly economically wasteful but problems of steric interference willarise which will actually serve to reduce the number of availableadsorption sites for the albumin.

There may be utilized between 0.1 to 5 grams of dye per 4 grams ofsupport phase. It is preferred however to utilize between 0.2 and 1 gramof dye per 4 grams of support phase. It should be noted however thatfrom a practical point of view the support phase is not usually measuredby weight but rather by volume of settled support phase in aqueoussuspension. Thus, where the support phase is sepharose, 4 grams ofsepharose would correspond substantially to 100 ml. of settledsepharose.

While the weight/volume ratio will of course vary with the chemicalnature of the support phase and the size of the granules, within thescope of generally available support phases of the size represented bysepharose of grades 2B, 4B, and 6B, the foregoing ratios aresufficiently close to provide a satisfactory range of operability.

In the preferred modification of the coupling procedure, the dye, thatis to say, between about 0.1 and 2 grams of dye, preferably between 0.2and 1 grams of dye, is dissolved or suspended in a small amount ofwater, between 10 and 50 ml. is generally suitable, and added to 100 ml.(measured as settled support phase of support phase, in water. Theamount of water to suspend the support phase is not critical; between100 ml. and 1000 ml. have been found suitable. The mixture is gentlyagitated and warmed to below the boiling point of water. A temperaturerange of 75° thru 95° C has been found satisfactory. The pH of thesolution is maintained slightly above pH 6, suitably between pH 6 and pH8 by the addition of either aqueous alkali or a buffer. The actualreaction time will depend upon the dilution of the dye, the temperature,and the agitation of the support phase. Nevertheless, the reaction cangenerally be considered to be complete in between from about 5 to about60 minutes. The coupled product (designated herein SP/CRD) is thenseparated from the aqueous phase, suitably by decantation and washed toremove any unreacted dye. It has been found suitable to wash thoroughlywith water at ambient temperature followed by a suitable salt, such asguanidine hydrochloride (5M) which will remove any unbound dye. The useof guanidine hydrochloride solution is not critical and other washes mayalso be used.

The thus purified SP/CRD is then suspended in a buffer, suitably abuffer of between a pH of 6.5 and 8.5. The albumin adsorptive capacityof the product thus produced will of course depend upon the dye/supportphase ratio. However, products produced in accordance with the foregoingprocedures will have an absorptive capacity of the order of 30milligrams of albumin per ml. of settled SP/CRD.

It should be borne in mind that different dyes will react at differentrates with different support phases. Furthermore care must be taken thatthe support phase does not become physically changed, i.e., by partialor complete melting, during the coupling process. For example, it shouldbe noted that while Cibacron Blue F-3GA reacts very rapidly withsepharose at 80° C, other dyes will couple slowly with ordinarysepharose at this temperature. While at 90° coupling occurs rapidly, theproduct produced is, although operative, not of optimum physicalcharacteristics. Thus, where higher reaction temperatures are required,it is desirable to utilize cross-linked support phases such ascross-linked sepharose.

As examples of other support phases which may be utilized in the processof the present invention, there may be mentioned polyacryamide gels, andacrylic resins containing amino groups, such as, for example,Amberlite®IRC 45.

Also included within the scope of the present invention are the Dextranderivatives of Cibacron Blue F3GA and Cibacron Brilliant Blue FBR-P. Inthese dyes the chloro group of the triazine moiety is replaced by an--O-- linkage to a Dextran molecule. The former when coupled withDextran 2000 yields Blue Dextran 2000 ®.

The foregoing coupling procedures for the formation of SP/CRD dependupon the reaction of a halo group on the triazine moiety with a labilegroup on the support phase. Such reaction is not feasible where thisgroup has been replaced by a --O--Dextran linkage. Nevertheless, wherethe CRD possesses a labile amino group such as, for example, the primaryamino group on the anthraquinone moiety of Cibacron Blue F3GA andCibacron Brilliant Blue FBR-P, coupling may be carried out by thecyanogen bromide method of Cuatracasas. In this procedure, the supportphase is activated by treatment with aqueous alkaline cyanogen bromideand the suitably buffered support phase reacted with the dye. In thisprocedure the Dextran/CRD in a similar buffer is then added to theactivated support phase and coupling allowed to proceed. The couplingreaction may take place at temperatures between 0° C and ambienttemperature, however, it is preferred to carry out the reaction atapproximately 4° C for approximately 24 hours. After elapse of thistime, the conjugate is successively washed with aqueous sodiumbicarbonate (pH approximately 9.5), concentrated aqueous urea(approximately 6M), water, and a high ionic strength buffer of pHbetween 6.5 and 8.5. It is preferred to utilize a tris-HCl/aqueoussodium chloride buffer. The sepharose/Dextran/CRD conjugate is thensuspended in fresh buffer and may be stored at approximately 4° C untilused.

In the practice of the albumin removal procedure it is found desirableto place the SP/CRD conjugate in a column in a high ionic strengthbuffer. The tris-HCl/sodium chloride buffer mentioned hereinabove hasbeen found especially suitable. The high ionic strength of the buffersystem is important for the operation of the procedure since it preventsthe SP/CRD from acting as a cationic exchange column. It has been foundsuitable to apply aproximately 2 ml. of albumin containing plasma toapproximately 10 ml. of settled SP/CRD. The plasma is then run throughthe column and the column washed with the aforementioned buffer. Elutionwas followed by uv adsorption of the eluate.

As stated heretofore the critical factor in this procedure is the ionicstrength of the buffer system. In order to be operative the buffershould have an ionic strength of at least 0.05M, no upper limit beingnecessary, at a pH of between 6.5 and 8.5, preferably a pH of about 8.0.

The thus obtained eluate is substantially albumin free and the remainingproteins may be isolated therefrom in methods known to the art.Examination and comparison of the initial plasma and the eluate showsthat in addition to the albumin only small amounts of other proteins,principally, lipoproteins are removed.

The column may be regenerated by breaking the (SP/CRD)/Albumin bond.Where preservation of the albumin is not required an aqueous solution ofurea or quanidine hydrochloride has been found suitable. The ureasolution must be at least 1M, while solutions of 3M have been used, anystrength up to saturation (ca 8M) is satisfactory. One column volume ofurea solution is sufficient to break the bonds, however, 1-3 columnvolumes are usually employed.

Where it is desired to preserve the albumin an ethanol/aqueous alkalimetal phosphate, suitable sodium phosphate solution is employed. Thepreferred volume ratio is 1:1 and the phosphate solution is between 0.05and 0.1M at pH of 2.0-3, suitably 0.075M at pH 2.4, measured prior tomixing.

It has also been found that albumin may be eluted in high purity usingan aqueous solution of thiocyanate together with, suitably the cationsof sodium, potassium and group 2A metals which provides sufficient ionicstrength. Especially suitable as cations are Na⁺ and K⁺. The ionicstrength of these eluents should be, preferably, at least 0.05M. Lowerconcentrations cause tailing. Higher concentrations, say 0.1M to 1.0Mbring a faster elution and are acceptable, these concentrations shouldnot be considered as limiting, nevertheless much higher concentrationsmay adversely affect the purity of the albumin.

It has been found desirable to buffer the eluent, suitably to a pHbetween pH 6.0 and 9, most preferably to a pH of about 8. This isachieved by adding a buffer such as sodium bicarbonate ortrishydrochloride to the eluent. An ionic strength of about 0.05-0.1Mtris hydrochloride in the eluent has been found suitable.

It has also been found that basic buffered alkanoic acids are suitableas eluents. Acids containing 2-10 carbon atoms may be employed, acidscontaining 8-10 carbon atoms are preferred. Octanoic acid is especiallysuitable as it is the stabilizing agent of choice for albumin. Utilizingalkanoic acids, especially octanoic acid as an eluent, there is madeavailable a stabilized solution of pure albumin which may, if desired,be concentrated under reduced pressure or lyophilized to provide purebuffered albumin.

Any buffer may be employed which will raise the pH of the solution to atleast 6.5, pH 7.5-9 being an operative range, and about pH 8.0 beingpreferred. Tris hydrochloride/aqueous sodiumchloride is especiallysuitable as a buffer.

It should be noted that the solubility of C₈ -C₁₀ alkanoic acids inwater is not high. Octanoic acid has a solubility of the order of 4mM.Thus the preferred mode of preparing the eluent is to prepare a buffersuch as tris hydrochloride (0.05-0.2M) suitably with 0.05-0.5M aqueoussodium chloride and add thereto as much of the alkanoic acid as willdissolve therein. Again, the solution should be buffered to at least pH6.5, pH 7.5 - pH 9, being the operative range and a pH of about 8.0being preferred.

Utilizing either method of column regeneration, the columns may berepeatedly utilized without loss of absorptive power.

                                      DYES UTILIZED AS CRD'S                      __________________________________________________________________________    CICN CI Name Trade Name (s)                                                                          Structural Formula                                     __________________________________________________________________________    17865                                                                              CI Reactive Orange                                                                    Cibacron Orange  G-E; Procion Brilliant Orange                                           ##STR3##                                              61210                                                                              CI Reactive Blue 5                                                                    Cibacron Brilliant Blue FBR-P                                                            ##STR4##                                              61211                                                                              CI Reactive Blue 2                                                                    Cibacron Blue F3G-A; Procion Blue HBS                                                    ##STR5##                                              18105                                                                              CI Reactive Red 4                                                                     Cibacron Brilliant Red 3B-A; Procion Brilliant                                           ##STR6##                                              26440                                                                              CI Reactive  Brown 1                                                                  Cibacron Brown 3GR-A; Procion Orange Brown HGS                                           ##STR7##                                              17910                                                                              CI Reactive Red 9                                                                     Cibacron Scarlet 2G; Procion Scarlet H3GS                                                ##STR8##                                                   CI Reactive                                                                           Cibacron Scarlet                                                                        --                                                          Red 16  4G-P                                                             18156                                                                              CI Reactive Red 12                                                                    Cibracon Brilliant Red B-A                                                               ##STR9##                                              18159                                                                              CI Reactive Red 3                                                                     --                                                                                       ##STR10##                                              --  --      Blue Dextran                                                                             ##STR11##                                              --  --      --                                                                                       ##STR12##                                             __________________________________________________________________________

EXAMPLE I

100 ml. of settled cross linked Sepharose®4B (Pharmacia Fine Chemicals,Inc) is suspended in 250 ml. of water. 1 gm. of Cibacron® Brilliant BlueFBR-P is dissolved in 25 ml. of water and the solution added to thesepharose suspension. The mixture is agitated gently and heated to 80° Cfor 45 minutes. The pH is held to at least pH 6 by addition of sodiumbicarbonate or tris hydrochloride. The reaction mixture is cooled toambient temperature, the supernatant liquid decanted and the solid phasewashed thoroughly, first with water (3 l.) then with aqueous guanidinehydrochlorde solution (2 l., 5M.) and finally suspended in a mixedbuffer of aqueous tris hydrochloride (0.05M) and aqueous sodium chloride(0.5M) (pH 8.0) to yield an SP/CRD conjugate of Sepharose 4B andCibacron Brilliant Blue FBR-P.

In accordance with the above procedures, there may be utilized packedsepharose polyacrylamide gel and Amberlite® IRC 45 to yield similarSP/CRD's.

In accordance with the procedures of the principle example, but using asCRD's, Procion Brilliant Orange HGRS, Procion Blue HBS, ProcionBrilliant Red H7B5, Procion Orange Brown HGS, Procion Scarlet H3GS,Cibacron Scarlet 4GP and Cibacron Brilliant Red BA, and as SP utilizinga cross-linked sepharose, polyacrylamide gel or a polyacrylic aminogroup containing resin similar SP/CRD's are obtained provided thetemperature of reaction is raised to 90°-95° C.

EXAMPLE II Preparation of Sepharose-Blue Dextran Conjugate

100 ml. of settled Sepharose 4B (Pharmacia Fine Chemicals Inc) istreated with an aqueous solution of cyanogen bromide (16g) at pH 11.0and 10° C for 5 hours. After completion of the reaction the supernatantliquid is removed by decantation and the sepharose agitated with sodiumbicarbonate buffer (0.1M, pH 9.5). The supernatant liquid is againremoved by decantation and Blue Dextran (1g) in 100 ml. of aqueoussodium bicarbonate, (0.1M, pH 9.5) is added. The reagents are allowed tostand for 24 hours at 4° C. The supernatant liquid is then removed bydecantation and the product washed successively with aqueous sodiumbicarbonate (4 l, 0.1M, pH 9.5), aqueous urea (4 l., 6M), water (4 l.)and a mixed buffer consisting of aqueous tris-hydrochloride (0.05M) andaqueous sodium chloride (0.5M) (4 l., pH 8.0). After removal of thesupernatant wash of this last buffer, the thus produced SP/CRD issuspended in a similar solution and, if desired, stored at 4° C.

EXAMPLE III Removal of Albumin from Plasma

A chromatographic column (1.0 × 20 cm) is charged with circa 10 ml. ofSP/CRD produced in accordance with Example II suspended in theaforementioned tris-hydrochloride/sodium chloride buffer. 2 ml. ofplasma is applied to the column and eluted with buffer until no furtherprotein was eluted. This is checked by ultra-violet examination of theeluate wherein A₂₈₀ is less than 0.020. The eluate is then reduced involume by diaflowing (utilizing an Amicon-UM10 membrane) to a volume of2 ml.

The procedure was controlled by running control samples through aSepharose 4B column which had not been conjugated with Blue Dextran. Thediaflowed eluate is then subjected to assay by cellulose acetatemembrane electrophoresis to yield the results set forth in Table I.

                  TABLE I                                                         ______________________________________                                        Recovery of Plasma Protein Fractions                                          After Passage Through Sepharose-Blue Dextran                                           Control  SBD-Treated                                                          Plasma  Plasma                                                       Fraction   (grams/100 ml.) % Recovery                                         ______________________________________                                        Albumin    4.10      0.16       4                                             Alpha-1    0.17      0.16      94                                             Alpha-2    0.72      0.60      84                                             Beta       0.67      0.65      96                                             Gamma      0.95      0.93      98                                             Total Protein                                                                            6.61      2.50      38                                             ______________________________________                                         Fractions were quantitatively assayed after cellulose acetate membrane        electrophoresis as described in text. Total protein was determined by the     method of Lowry, et al, (J. Biol. Chem. 193, 265 (1951)).                

EXAMPLE IV Removal of Albumin from Plasma

A chromatographic column (1.0 × 20 cm) is charged with circa 10 ml. ofSP/CRD produced in accordance with Example I utilizing as SP, crosslinked Sepharose 4B and as CRD, Procion Brilliant Red H7BS, suspended inthe aforementioned tris-hydrochloride/sodium chloride buffer. 2 ml ofplasma is applied to the column and eluted with buffer until no furtherprotein was eluted. This is checked by ultraviolet examination of theeluate wherein A₂₈₀ is less than 0.020. The eluate is then theaforementioned tris-sodium chloride buffer.

EXAMPLE V Removal of Albumin from Plasma

A chromatographic column (5 × 100 cm) is charged with circa 1000 ml ofSP/CRD produced in accordance with Example I utilizing as SP, crosslinked Sepharose 4B and as CRD, Cibracrom Blue F3GA, suspended in theaforementioned tris-hydrochloride/sodium chloride buffer. 600 ml ofplasma is applied to the column and eluted with buffer until no furtherprotein was eluted. This is checked by ultraviolet examination of theeluate wherein A₂₈₀ is less than 0.020. The eluate is then theaforementioned tris-sodium chloride buffer.

EXAMPLE VI Regeneration of Column with Albumin Recovery

The albumin containing column of Example V is washed with an aqueouseluent comprising sodium thiocyanate (0.3M) and tris-hydrochloride(0.05M, pH8) until no further albumin is shown to be present in theeluate by optical density monitoring.

The eluate is then dialyzed against distilled water using an AmiconDC-52 hollow fiber. The dialysis is continued until the wash water has aconductivity of less than 50 micro-mho and is negative in the ferricchloride test indicating removal of the thiocyanate.

The dialysis residue is then concentrated and lyophilized to yieldalbumin of purity exceeding 98%.

In accordance with the above procedure any of the SP/CRDS produced inaccordance with Examples I and II may be employed. In accordance withthe above procedures in place of sodium thiocyanate, the cation may beammonium, potassium or any group 2A metal giving rise to a sufficientsolubility to reach the required concentration range.

EXAMPLE VII Column Regeneration

The column which was eluted in accordance with Example VI is preparedfor reuse by washing with saline tris-hydrochloride buffer (N_(a) Cl0.05M, Tris-HCl 0.05M, pH 8.0).

We claim:
 1. A method of obtaining albumin from substantially cell freeaqueous fluids containing the same comprising the steps ofa. suspendinga composition of the general formula (SP)_(x) (CRD) wherein CRD is acolor reactive dye of the general formula ##STR13## wherein R₃ ishydrogen, alkyl, phenyl, alkoxy phenyl, alkyl phenyl or ##STR14##wherein R₁ is hydrogen, an alkali metal, or an alkaline earth metal, R₂is halo or --O--dextran,Z₁ is two hydrogen atoms, or --CH═CH--CH═CH-- toform a naphthyl nucleus with the phenyl nucleus to which it is attached,Z₂ is one bond of a nitrogen-nitrogen bond or a hydrogen attached to thenitrogen shown only, Q is substituted or unsubstituted aryl where Z₂ ishydrogen, or Q is substituted or unsubstituted aryl-N═ where Z₂ is onebond of a double bond, m is 1 or 0, n is 1 or 2, provided that thesubstituent groups aforesaid are other than halo or heavy metal, andwherein SP is a solid support phase selected from the group consistingof agarose, polyacrylamide and acrylic resin containing amino groups,provided that where the CRD, R₂ is halo, the SP- linkage in the (SP)_(x)(CRD) is to the R₂ bearing position of the triazine moiety, and where inthe CRD R₂ is O-dextran, the SP- linkage in the (SP)_(x) (CRD) is to theQ moiety, and x is expressed as a weight ratio between 0.8 and 40, in abuffer of at least 0.05 M molarity at a pH of between 6.5 and 8.5; b.contacting said suspension with said albumin containing fluid to providea suspension having albumin absorbed upon the solid phase thereof; c.separating the solid phase-albumin adsorbate from the aqueous phase bytreating the solid phase-albumin adsorbate with an aqueous ionic eluentwherein the anion is thiocyanate.
 2. A method of claim 1 wherein SP/CRDhas the general formula ##STR15## wherein x, Q, Z₁, Z₂, and R₁ aredefined as in claim
 1. 3. A method of claim 2 wherein SP is sepharose.4. A method of claim 2 wherein SP is a polyacrylamide gel.
 5. A methodof claim 2 wherein SP is an acrylic resin containing amine groups.
 6. Amethod of claim 1 wherein SP/CRD has the general formula ##STR16## x,and R₁ are as defined in claim
 1. 7. A method of claim 6 wherein SP issepharose.
 8. A method of claim 6 wherein SP is a polyacrylamide gel. 9.A method of claim 6 wherein SP is an acrylic resin containing aminogroups.
 10. A process according to claim 3 wherein CRD is Procion BlueHBS or Cibacron Brilliant Blue FBR-P.
 11. A process according to claim 3wherein CRD is Procion Orange Brown HGS, Procion Brilliant Red H7BS,Procion Scarlet H3GS, Procion Brilliant Orange HGRS.
 12. A methodaccording to claim 1 wherein the eluent comprises a cation of a metal ofgroup 2A of the periodic table, sodium or potassium and also a basicbuffer.
 13. A method of claim 1 wherein the thiocyanate has aconcentration of between 0.1M and 1.0M.
 14. A method in accordance withclaim 12 wherein the eluent is an aqueous solution of sodium thiocyanateand a basic buffer.
 15. A process of claim 1 additionally comprising thestep of isolating the albumin by dialysis against water.
 16. A processof claim 15 wherein the dialysis is carried out against distilled waterto a conductivity below 50μmhos, said water giving a negative result inthe ferric chloride test for thiocyanate.
 17. A process of claim 16further comprising removal of water from the dialysate containing thealbumin.
 18. A process of claim 17 wherein the water is removed byconcentration and lyophilization.
 19. A method of removing albumin fromaqueous fluids containing the same comprisingi. the method of claim 1ii. treating the thus produced solid phase-albumin adsorbate with anaqueous eluent comprising thiocyanate anions and cations selected fromthe group consisting of cations of groups 2A metals sodium andpotassium, said eluent further comprising a basic buffer, iii.separating the thus formed albumin free solid phase from the liquidphase, iv. utilizing said albumin free solid phase in step (a) of claim1, v. repeating steps (i) thru (iv) hereof.